Project Summary Adjuvants are essential components of vaccines, and function to enhance the immune response to a co-administered antigen. Adjuvants are a primary focus of modern vaccine development due to the fact that despite decades of research only two adjuvants, alum and monophosphoryl lipid A (MPLA), are clinically approved for human use. While these adjuvants are effective in stimulating immune responses for some vaccines, their ability to potentiate immune responses against weakly immunogenic haptens or highly diverse antigens has achieved mixed results. For example, clinical translation of vaccines for hepatitis C virus (HCV) has been stunted by the remarkable diversity of the virus and the virus? immune evasion mechanisms. To address these shortcomings and those of other vaccines, we have undertaken a program to discover new, broadly-applicable adjuvant platforms. Of late, the Janda Laboratory has investigated 4-((2-formyl-3-hydroxyphenoxy)methyl)benzoic acid (tucaresol) as a synthetically-tractable adjuvant. Recently, we reported the first liposomal formulation of tucaresol, termed L(LT1). We used this adjuvant in vaccines for HCV, herpes simplex virus 2, and methamphetamine, and demonstrated high antibody titers in mouse models. Despite our initial successes, our understanding of how L(LT1) stimulates the immune system remains limited. In this proposal, we will elucidate the mechanism of L(LT1) immunopotentiation. Our approach will include investigating the induction of Th-cell proliferation, cytokine production, and expression of co-stimulatory cell surface markers (Aim 1) and evaluating a series of L(LT1) analogues in rodents for their ability to stimulate an immune response after HCV vaccination (Aim 2). In Aim 2, a previously-constructed SAR series will dissect how tucaresol?s chemical makeup plays a role in immunostimulation. The proposed work will help to achieve our long-term goal of delivering a clinically- relevant adjuvant platform with broad applicability to many vaccines. The work proposed here is innovative and impactful because it marks the first detailed, mechanistic study of immunopotentiation by L(LT1). These pioneering studies will have a lasting impact because they provide a foundation for the future development of L(LT1) analogues as next-generation vaccine adjuvants. This work is directly aligned with the mission of NIAID to support research aimed at improving the treatment and prevention of infectious diseases.